Inflammation is a localized protective response elicited by tissues in response to injury, infection, or tissue destruction resulting in the destruction of the infectious or injurious agent and isolation of the injured tissue. A typical inflammatory response proceeds as follows: recognition of an antigen as foreign or recognition of tissue damage, synthesis and release of soluble inflammatory mediators, recruitment of inflammatory cells to the site of infection or tissue damage, destruction and removal of the invading organism or damaged tissue, and deactivation of the system once the invading organism or damage has been resolved. In many human diseases with an inflammatory component, the normal, homeostatic mechanisms which attenuate the inflammatory responses are defective, resulting in damage and destruction of normal tissue.
Cell-cell interactions are involved in the activation of the immune response at each of the stages described above. One of the earliest detectable events in a normal inflammatory response is adhesion of leukocytes to the vascular endothelium, followed by migration of leukocytes out of the vasculature to the site of infection or injury. The adhesion of these leukocytes, or white blood cells, to vascular endothelium is an obligate step in the migration out of the vasculature (Harlan, J. M., Blood 1985, 65, 513-525). This response is mediated by the interaction of adhesion molecules expressed on the cell surface of leukocytes and vascular endothelial cells.
Very late activating antigen-4 (also called VLA-4, α4β1 or CD49d/CD29) is an integrin expressed in the surface of lymphocytes, monocytes, macrophages, mast cells, basophils and eosinophils. It is a heterodimeric adhesion receptor which is composed of noncovalently linked α4 and β1 subunits and serves to mediate leukocyte adhesion to vascular cell adhesion molecule-1 (VCAM-1) which is expressed on cytokine-stimulated endothelial cells. This interaction between VCAM-1 and VLA-4 contributes to leukocyte extravasation in acute and chronic inflammatory conditions including multiple sclerosis (MS), rheumatoid arthritis, asthma, psoriasis and allergy.
The α4 integrin subunit can also heterodimerize with a β7 integrin chain to form integrin α4β7 which is known as a mucosal homing receptor because its primary ligand is the mucosal vascular adhesion molecule MadCAM-1. Integrin α4β7 identifies a subset of memory T cells with a tropism for the intestinal tract, whereas integrin α4 μl (VLA-4) is constitutively expressed on most mononuclear leukocytes, but not on circulating neutrophils. The interaction of VCAM-1 with VLA-4 suggests that VLA-4 is a potential therapeutic target for inflammatory diseases, including many respiratory conditions, including, for example, asthma and bronchitis (Kassner, P. D., et al, Adv. Exp. Med. Biol. 1992, 323, 163-170).
Asthma is an inflammatory disease associated with eosinophil infiltration into the lung. VLA-4 is expressed on eosinophils. Metzger, W. J. (Springer Semin. Immunopathol. 1995, 16, 467-478) used a rabbit model of asthma to demonstrate that both an anti-VLA-4 antibody and a CS-1 peptide could reduce eosinophil infiltration into the lung and reduce the development of asthma.
While steroids and other antiinflammatory drugs are effective in treating inflammatory diseases and conditions, long-term usage often leads to side effects such as increased risk of infection caused by impairment of phagocytic leukocyte migration and function. There is some concern that inhibition of the function of the β1 integrin chain may be associated with increased susceptibility to infections, as demonstrated by a β1 (also called CD18) monoclonal antibody in rabbits (Foster, C. A., 1996, J. Allergy Clin. Immunol., 98, 270-277). It is believed that selective inhibition of the α4 chain may be a more desirable approach. Inhibition of the α4 chain is believed likely to reduce levels of the VLA-4 heterodimer as well as the α4β7 heterodimer.
Potential therapeutic interventions targeting VLA-4 include monoclonal antibodies and peptide antagonists. Antibodies specific for VLA-4 have been effective in attenuating allergen-driven airway inflammation and hyperresponsiveness in several experimental models of asthma, including the mouse. Leger, O. J. P. et al. (Human Antibodies, 1997, 8, 3-16) describe a monoclonal antibody against VLA-4 that is in phase III clinical trials for multiple sclerosis. CS-1 peptide antagonists have been described by Jackson, D. Y., et al. (J. Med. Chem. 1997, 40, 3359-3369). Hayashi et al. (Cell Struct. Funct. 1991, 16, 241-249) have used a vector expressing RNA complementary to chicken integrin β1 to reduce integrin β1 expression, resulting in altered cell attachment and shape.
Antisense oligonucleotides (“ASOs”) targeted to various integrins have been used as tools to dissect the functional interactions of integrins in complex settings. Lallier and Bronner-Fraser (Science, 1993, 259, 692-695) have used phosphorothioate oligonucleotides targeted to conserved and nonconserved regions of chick β1, human α4, rat α1 and human β5 integrins to determine the effects of these integrins on cell attachment. These same oligonucleotides were also injected into cranial neural crest migratory pathways in avian embryos, and it was demonstrated that those oligonucleotides that inhibited cell attachment in vitro also caused neural crest and/or neural tube abnormalities in vivo (Kit et al., Devel. Biol. 1996, 179, 91-101).
EP patent application 688 784 (Carolus et al.) discloses 3′ derivatized oligonucleotide analogs, including one sequence targeted to the β1 subunit of VLA-4.
U.S. Pat. Nos. 5,968,826 and 6,258,790 (Bennett et al.) describes modulating integrin α4 expression through the use of antisense oligonucleotides targeted to nucleic acids encoding integrin α4. U.S. Pat. No. 5,968,826 discloses that such antisense oligonucleotides can be used to treat a large variety of inflammatory diseases associated with VLA-4 expression, including asthma. According to this document, in general, a dosage of from 0.01 μg to 100 g of antisense oligonucleotide per kg of body weight, which may be given once or more times daily, weekly, monthly or yearly, or even every 2 to 20 years, may be used to treat the inflammatory diseases. The range of dosages exemplified for the various inflammatory diseases is from 0.01 mg to 20 mg of antisense oligonucleotide per kg body weight. Example 30 describes the prophetic use of antisense oligonucleotides in a murine model for asthma in which mice are injected intravenously with 1 mg/kg to 5 mg/kg doses of antisense oligonucleotides to integrin α4.